Plant Physiol. Illumina
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Plant Physiology 65:465-468 (1980)
© 1980 American Society of Plant Biologists

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Simultaneous Kinetic Analysis of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activities 1

Samuel S. Kent2 and Joseph D. Young

Department of Botany and Range Science, Brigham Young University, Provo, Utah 84602

An assay was developed for simultaneous kinetic analysis of the activities of the bifunctional plant enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase [EC 4.1.1.39]. [1-14C,5-3H]Ribulose 1,5-bisphosphate (RuBP) was used as the labeled substrate. Tritium enrichment of the doubly labeled 3-phosphoglycerate (3-PGA) product, common to both enzyme activities, may be used to calculate Vc/Vo ratios from the expression A/(B-A) where A and B represent the 3H/14C isotope ratios of doubly labeled RuBP and 3-PGA, and Vc and Vo represent the activities of carboxylase and oxygenase, respectively. Doubly labeled substrate was synthesized from [2-14C]glucose and [6-3H]glucose using the enzymes of the pentose phosphate pathway coupled with phosphoribulokinase.

The kinetic properties of a commercial preparation of fully activated spinach carboxylase were studied under approximated physiological conditions of 20% O2 (252 micromolar), 295 µl/l CO2 (10 micromolar), 25 C, and pH 8.19. The Vc/Vo ratio was, within experimental error, constant at 30 seconds and 1 minute. This double label assay method may be used to calculate Vc/Vo ratios for the Laing-Ogren-Hageman equation, Vc/Vo = (VcKo/VoKc) ([CO2]/[O2]) where Vc and Vo represent Vmax, and Kc and Ko represent Michaelis constants for the carboxylase and oxygenase activities, respectively.

2 Present address: Department of Chemistry, Lyndon State College, Lyndonville, Vermont 05851. To whom reprints requests should be sent.

1 This work was supported by Brigham Young University Professional Development Fund 115-76-411.






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ASPB Publications PLANT PHYSIOLOGY® THE PLANT CELL
Copyright © 1980 by the American Society of Plant Biologists